Our long term goal is to identify mechanisms of cellular long chain free fatty acid (LCFFA) uptake and their possible roles in disease. Our earlier work helped prove that the major LCFFA uptake process under physiologic conditions is facilitated transport showed altered tissue specific regulation of this process in adipocytes in a rat model of obesity and type 2 diabetes and hepatocytes during exposure to ethanol and isolated the first plasma membrane LCFFA transporter) which, surprisingly, proved identical to mitochondrial aspartate aminotransferase (mAspAT). During the 1998-2002 funding period, we showed: [1] that tissue-specific up-regulation of adipocyte LCFFA uptake was characteristic of both genetic and dietary obesity in several additional rodent models and adipocytes from obese human subjects; [2] that the extent of up-regulation was closely correlated with the rate of weight gain in a rat model of dietary obesity; and [3] that up-regulation or down-regulation of facilitated LCFFA uptake in adipocytes preceded weight gain or weight loss, respectively, in several experimental settings, suggesting that regulation of adipocyte LCFFA uptake is an important control point for body adiposity. In one such study, administration of leptin to hyperinsulinemic ob/ob mice led sequentially to a rapid fall in plasma insulin levels, a progressive decrease in the rate of facilitated LCFFA uptake by adipocytes, decreased food intake, weight loss, and finally, a decrease in the size of adipocytes and of passive LCFFA uptake. These data suggest that insulin is an up-regulator and leptin a downregulator of adipocyte LCFFA uptake, but leave open the question of whether the leptin effect is direct or is mediated by its influence on insulin secretion and function. The current application seeks to build on these observations. SPECIFIC AIM 1 seeks to confirm the relationship between the extent of up-regulation of uptake and the rate of weight gain, and to verify that the observed changes in adipocyte LCFFA uptake result in changes in nutrient partitioning in the Osborne-Mendel rat, which is sensitive to diet-induced obesity. SPECIFIC AIM 2 is to clarify the roles of insulin and leptin in respectively up- and down-regulating adipocyte LCFFA uptake. In studies proposed for SPECIFIC AIM 3 we will use adipocyte culture systems to examine the effects of hormones and other potential regulators of adipocyte LCFFA uptake, and explore the effects of RNA silencing of putative LCFFA transporters on LCFFA uptake and triglycerides accumulation. Finally, SPECIFIC AIM 4 will characterize depotspecific differences in LCFFA uptake in freshly isolated human adipocytes removed at surgery, and will also determine the effects of massive weight loss resulting from bariatric surgical interventions on these transport processes. Since tissue-specific up-regulation of LCFFA uptake characterizes obesity, understanding the regulatory mechanisms may have therapeutic implications.